While molecular machineries involved in membrane fusion/fission have been dissected, regulation of fusion/fission dynamics remains poorly understood. We uncovered ENV7 as a novel gene involved in vacuolar events and have established it as an ortholog of STK16-related kinases. We have shown that Env7 protein is a palmitoylated vacuolar membrane kinase that negatively regulates vacuolar membrane fusion during budding and hyperosmotic stress. Additionally, we have established that Env7 phosphorylation in vivo is dependent on YCK3, a gene encoding another vacuole membrane kinase with a non-redundant function in fusion regulation. Deletion of both genes results in severe morphological defects and compromised growth. Thus, Env7-mediated membrane flux regulation appears to be part of a novel kinase cascade affecting global cell fitness. Based on our findings to date, we propose a model for regulation of Env7 function and hypothesize that 1) Env7-mediated vacuole fusion/fission is regulated through its phosphorylation by Yck3 and 2) Env7 substrate(s) include component(s) of the vacuolar fusion/fission machinery. Here, we propose to test various aspects of the proposed model through a systematic multifaceted approach that includes live cell imaging, biochemical analyses, in vivo and in vitro fusion assays, and global proteomic studies. If successful, this project will establish Env7 as a node in a novel kinase signaling cascade involved in regulation of membrane fusion/fission dynamics. This will be a leap forward in organelle biogenesis, membrane dynamics, and signaling. The proposed experiments also represent the first studies on regulation and cellular substrates of any STK16-related kinase. Inherent in this SCORE proposal, is the aim to develop the depth and research productivity of the PI within the highly active fields of membrane fusion and kinase cascades, while continuing the productive research training of undergraduate and Master's students within a comprehensive Hispanic Serving Institute. Lysosomal defects have been associated with Lysosomal Storage Diseases and neurodegeneration. Defects specifically in lysosomal fusion dynamics have recently emerged as the possible underlying mechanism in pathologies associated with both Lysosomal Storage Diseases and Alzheimer's disease.